Over the next year we plan to continue study of the acid-resistant nucleating factor in micropuncture fluid. Inasmuch as we have evidence that the fluid comes from between cartilage cells, we have perhaps the strongest approach to obtain a factor nearest to the native state so far available. For all other methodologies there is either doubt about nucleating capacity or some doubt as to whether artificial vesicles are made during the preparation. We will study the proteins and matrix vesicle membranes by SDS gels in two dimensions, and try to make a comparison with upper 1/5 puncture fluid profiles at microscale on enzymically separated vesicles, a project already underway. A test to identify whether a calcium binding phospholipid complex is present as the nucleating agent (a lipoprotein less likely) or a phospholipid protein complex is present will be investigated. The density of the material in the upper 1/5 must be extremely low and the possibility that these are vesicle membranes somewhat deleted of proten will also be examined. Further studies will be made on the inorganic phosphate generating capacity of Cfl as well as the depression of this function by EHDP is so far suspected in early experiments. The proteoglycan nucleating agent inhibitor will also be studies in respect to a possible role of lysozyme. Finally, the calcium binding properties of the nucleational agent in the new diffusion cell will be studied. Attempts will be made to determine the relationship of inhibition of various phosphatases to function of the nucleational sites.